Background[unreadable] [unreadable] During the last several years our laboratory has studied five different dominantly-inherited autoinflammatory disorders. The first of these illnesses is the TNF receptor-associated periodic syndrome (TRAPS), which is characterized by oftentimes prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. In 1999 our group identified the first six mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in seven families with dominantly-inherited recurrent fevers, and therefore proposed the name TRAPS for this clinical condition. Initial mechanistic studies indicated a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired homeostasis of the immune response. [unreadable] [unreadable] In 2002 we and a French group independently discovered dominantly-inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), in about 50% of patients with a disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. Two phenotypically milder conditions, familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), are caused by mutations in the same gene. CIAS1 encodes a protein, cryopyrin, that participates in a macromolecular complex called the inflammasome to regulate the activation of interleukin-1 (IL-1) beta.[unreadable] [unreadable] A fifth dominantly-inherited autoinflammatory disorder, denoted the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), is caused by mutations in a protein known as proline serine threonine phosphatase interacting protein (PSTPIP1). PAPA is characterized by episodes of mono- or pauciarticular sterile pyogenic arthritis, which can be destructive if not treated, formation of open, purulent ulcers of the skin (pyoderma gangrenosum), and severe cystic acne. In 2003 our group discovered that PSTPIP1 binds pyrin, the protein mutated in familial Mediterranean fever (FMF), and that disease-associated mutations in PSTPIP1 lead to more avid binding to pyrin, and increased IL-1 beta activation, relative to healthy controls.[unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Functional studies of mutant TNFRSF1A molecules: In collaboration with the laboratory of Dr. Richard Siegel, we have continued analyses of the functional consequences of TRAPS-associated TNFRSF1A mutations. Studies performed during previous reporting periods have demonstrated that the aforementioned defect in TNFR ectodomain cleavage is unlikely to explain all or most of the autoinflammatory phenotype in this disorder, and in transfection systems the mutant receptors have been found to exhibit diminished binding to TNF and abnormal intracellular trafficking, with retention in the endoplasmic reticulum (ER). A number of studies during the present reporting period have focused on peripheral blood mononuclear cells (PBMCs) from TRAPS patients and controls. We found that although TNFRSF1A mRNA levels are similar between patients and controls, the amount of protein in whole cell lysates is significantly higher. Cycloheximide studies indicate a prolonged TNFRSF1A protein half-life in TRAPS, consistent with the finding of ER retention in cell lines. [unreadable] [unreadable] We also found increased phosphorylated JNK in unstimulated TRAPS PBMCs. To probe the sensitivity of TRAPS PBMCs to stimulation, we studied cytokine secretion. PBMCs from 13 TRAPS patients without fever and 13 controls were incubated for 24 hours with or without lipopolysaccharide (LPS) and cytokine concentrations were measured in supernatants. TRAPS PBMCs did not show spontaneous secretion of proinflammatory cytokines, including TNF, IL-1beta, IL-6, and IL-8. However, at low concentrations of LPS, TRAPS PBMCs secreted significantly more IL-1beta, TNF, and IL-6 than control PBMCs. This increased cytokine response was especially prominent in TRAPS patients with normal serum C-reactive protein levels at the time of phlebotomy.[unreadable] [unreadable] Gene expression profiling in NOMID: In 16 of 18 patients with NOMID who were treated with the IL-1 receptor antagonist anakinra, we performed gene expression profiling of mRNA from PBMCs with Affymetrix U133A 2.0 microarrays, and selected transcripts were validated by quantitative real-time PCR. Among the validated genes were a number with known immunologic function, including STAT1, SOCS3, MMP9, SOD2, IFNGR1, and BCL6. We also found several genes that are downregulated by anakinra that do not have a well-characterized role in inflammation. One such gene is SNCA, encoding alpha-synuclein, the protein product of which is mutated in some forms of familial Parkinsons disease, and which we have found to be expressed in human monocytes, granulocytes, and lymphcytes from healthy controls. [unreadable] [unreadable] Studies of IL-1 inhibition in the cryopyrinopathies: In collaboration with Dr. Raphaela Goldbach-Mansky, we continue therapeutic trials of anakinra and a longer-acting IL-1 inhibitor manufactured by Regeneron pharmaceuticals and denoted IL-1 Trap. These studies are more thoroughly presented under project Z01 AR041138-05 OCD.[unreadable] [unreadable] Generation of new animal models: We are currently in the process of developing new animal models for the cryopyrinopathies and PAPA syndrome. In collaboration with Dr. Hal Hoffman, we are developing models for FCAS (CIAS1 L3533P), MWS (A352V), and NOMID (D303N, Y570C). We are also generating PSTPIP1 knockout mice, and the PSTPIP1 E250Q PAPA knockin mouse.[unreadable] [unreadable] Development of a resequencing chip for mutational screening of undiagnosed patients: With Affymetrix, we have developed a resequencing chip to interrogate approximately 300 kb of genomic sequence in patients with currently genetically uncharacterized periodic fevers. There are 91 genes represented on the chip, including those known to cause autoinflammatory disease, genes encoding inflammasome components, cytokine and cytokine receptor genes, and genes encoding NF-kappa B components, various signaling molecules, myeloid proteins, and regulators of apoptosis. [unreadable] [unreadable] Conclusions and Significance[unreadable] [unreadable] Studies on the functional consequences of TNFRSF1A mutations suggest a more complex pathophysiologic model of TRAPS than previously appreciated, in which constitutive cellular activation may play a prominent role. Studies of patients undergoing treatment for NOMID are likely to reveal new inflammatory genes and regulators. During the next year we plan to continue collaborative studies of TNF receptor cell biology and NOMID treatment, to begin screening patients with undiagnosed autoinflammatory diseases with our newly developed resequencing chip, and to begin phenotypic studies on CIAS1 and PSTPIP1 mice as appropriate backcross lines are bred.